The present invention relates to an improved automatic measurement method of glycohemoglobin based on the principle of high pressure liquid chromatography and to an improved sample injection valve used for a high pressure liquid chromatograph.
Glycohemoglobin (HbA.sub.1) attaching glucose to hemoglobin is often found in diabetes patients, and particularly HbA.sub.1c has been an important measurement item as an index for a health screening such as a medical checkup or for a long-term control of diabetes. Because, HbA.sub.1c exists most abundantly in glycohemoglobin (HbA.sub.1) and the increase by diabetes is much more than in the other component, and moreover the value of HbA.sub.1c shows a significant correlation with an average blood sugar level in hunger for past 1.about.3 months.
Glycohemoglobin includes HbA.sub.1a and HbA.sub.1b besides HbA.sub.1c, and these fractions are measured by colorimetry, electrophoresis, minicolumn method, or high pressure liquid chromatography. Among them, in the area of clinical tests, recently high pressure liquid chromatography (HPLC) has been prevalent in view of required time and separation.
A so-called stable type in HbA.sub.1c shows a significant correlation with a past blood sugar level and besides a labile type does not so. The rate of the latter is said to be 10.about.15% in all HbA.sub.1c in hunger on healthy human. In this labile HbA.sub.1c, the N end of .beta.-chain of hemoglobin and the reductive end of glucose form a reversible Shiff-base combination, which generates and degrades in a relatively short time depending on blood sugar levels. Therefore, it exists in diabetes patients more than in healthy, sometimes being 10.about.20% to all HbA.sub.1c. After meal it exists more than in hunger, being effected largely by the condition in collecting blood.
Stable HbA.sub.1c is generated from labile HbA.sub.1c gradually, continually, and irreversibly, reflecting the past long-term blood sugar levels. Thus, the separate measurement of the stable type only is desirable. However, both of the stable and labile types closely resemble structurally, being considerably difficult to separate by liquid chromatography.
As a method against this, the elevation of separation has been conducted by using a long high-separation column. Though this method has a merit that the denaturalization of glycohemoglobin by chemical treatment may not occur, the analyzing time of ten and several min or more is necessary to gain a good separation, insufficiently dealing with the increase of samples or urgent measurement. Moreover, this type of column is long leading to the apparatus of large type and high cost.
As another method for separately measuring the stable HbA.sub.1c, there is a method wherein the labile HbA.sub.1c is removed by degrading chemically in pretreatment. This is based on that the temporary combination (Shiff base) of labile HbA.sub.1c and glucose is easy to degrade. For instance, washed red cells are incubated in isotonic phosphoric acid buffer solution (37.degree. C., 4 hrs) or in physiological saline solution (room temperature, 14 hrs) to release glucose from labile HbA.sub.1c.
Or, there is also a method wherein whole blood added a hemolyzing reagent is incubated at 35.degree. C. for ten and several hrs. It reduces the level of labile HbA.sub.1c by hemolyzing and diluting a sample, particularly having a large effect in the acidic area below pH 6, a fast reaction speed, and a large effect at an elevated temperature. Further, the addition of the reagent for removing labile HbA.sub.1c containing boric acid on the market which has been used for minicolumn method enlarges the effect.
However, the pretreatment requires time, and together with the degradation of labile HbA.sub.1c, the degradation and change of other glycohemoglobin and pure hemoglobin (HbA.sub.o) proceed also at the same time.
The hemolyzing type has had a defect that the quantity of stable HbA.sub.1c varies with the time elapsed after hemolyzing and the temperature experienced till measurement. Particularly, in the case of the automatic measurement of a number of samples, there has been a disadvantage that the dilution of blood sample by the hemolyzing liquid containing a degrading reagent changes the sample measured later in samples on standby by excessive proceeding of reaction because of longer time elapsed.